A total of 200 fish, 100 Oreochromis niloticus weighing (60-300 g) and 100 Tilapia zillii  weighing (40-70 g) were collected randomly from different sites in Ismailia Governorate Egypt, during the period from October 2015 to September 2016. The clinical signs and postmortem lesions in naturally infected fishes were represented as hemorrhages on external body surface, hemorrhagic ulcer and congestion in most internal organs. The total prevalence of isolated Ps. aeruginosa in O. niloticus was 40% with the highest prevalence in winter 60% while the lowest prevalence was in summer 24%. The highest intensity was from liver 75% while the lowest was from gills 20%. On the other hand, prevalence in T. zillii was 22% with the highest in winter 36% while the lowest prevalence was in summer 8%. The highest intensity was from liver 72.7% while the lowest was from gills 13.6%. PCR is sensitive, rapid and specific method to detect resistant genes (MexA at 293 bp and MexB at 244 bp) and 16S rRNA gene at 530 bp in the selected isolates of Ps. aeruginosa. The antibiogramme of Ps. aeruginosa isolates showed high sensitivity to ampicillin, cefalexin and trimethoprim/sulfamethoxazole while they were resistant to amoxicillin and tobramycin. Challenge test revealed that the mortality rate in O. niloticus and T. zillii with Ps. aeruginosa by I/P route representing 80 and 50% of the total fishes, respectively. It was concluded that, O. niloticus is more susceptible to Pseudomonas septicemiathan T. zillii in both natural and experimental infection